Exosome‐derived long non‐coding RNA ADAMTS9‐AS2 suppresses progression of oral submucous fibrosis via AKT signalling pathway

Abstract Oral submucosal fibrosis (OSF) is one of the pre‐cancerous lesions of oral squamous cell carcinoma (OSCC). Its malignant rate is increasing, but the mechanism of malignancy is not clear. We previously have elucidated the long non‐coding RNA (lncRNA) expression profile during OSF progression at the genome‐wide level. However, the role of lncRNA ADAMTS9‐AS2 in OSF progression via extracellular communication remains unclear. lncRNA ADAMTS9‐AS2 is down‐regulated in OSCC tissues compared with OSF and normal mucous tissues. Low ADAMTS9‐AS2 expression is associated with poor overall survival. ADAMTS9‐AS2 is frequently methylated in OSCC tissues, but not in normal oral mucous and OSF tissues, suggesting tumour‐specific methylation. Functional studies reveal that exosomal ADAMTS9‐AS2 suppresses OSCC cell growth, migration and invasion in vitro. Mechanistically, exosomal ADAMTS9‐AS2 inhibits AKT signalling pathway and regulates epithelial‐mesenchymal transition markers. Through profiling miRNA expression profile regulated by exosomal ADAMTS9‐AS2, significantly enriched pathways include metabolic pathway, PI3K‐Akt signalling pathway and pathways in cancer, indicating that exosomal ADAMTS9‐AS2 exerts its functions through interacting with miRNAs during OSF progression. Thus, our findings highlight the crucial role of ADAMTS9‐AS2 in the cell microenvironment during OSF carcinogenesis, which is expected to become a marker for early diagnosis of OSCC.

geographical pathogenesis, and the first case was reported in 1985 in mainland China. 3 The pathology of OSF can be divided into the early, middle and advanced stages, which are possible processes to OSF carcinogenesis. Recent studies have shown that the cancerous rate of OSF is increasing by reaching 3%-19%. 4,5 Thus, identification of key molecular events in the malignant progression of OSF will help improve the early diagnosis and prevention of OSCC.
Exosomes are extracellular messengers that transport and exchange substances between tumour cells and microenvironment. 6,7 Long non-coding RNA (lncRNAs) can be packaged into exosomes and act as messengers for intercellular communication, participating in the regulation of cell microenvironment. Deregulation of exosomal lncRNA affects the tumour microenvironment such as angiogenesis, metastasis and drug resistance and thus contributes to tumorigenesis. 8 Our preliminary work has interpreted the lncRNA expression profile during the malignant evolution of normal oral mucosa-OSF-OSCC at the genome-wide level for the first time. 9 We found that the key signalling pathways involved in OSF carcinogenesis are chemokine and cytokine-mediated inflammatory signalling pathway, Wnt signalling pathway and angiogenesis signalling pathway, which are closely related to alterations in the tumour microenvironment.
The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family promotes or inhibits the tumorigenic potential of tumour cells through alterations in tumour microenvironment. 10 ADAMTS family proteins are involved in a variety of biological processes, including fibrosis, angiogenesis, cell invasion and metastasis, and tumorigenesis. 11 The role of ADAMTS family proteins in fibrosis of the myocardium, 12,13 liver 13-15 and lung 13,16,17 has been well studied. Moreover, genetic and epigenetic alterations (mutation, CpG methylation) of ADAMTS family proteins in multiple tumours including head and neck cancer indicate their direct roles to cancer initiation and progression through exerting oncogenic or anti-tumour effects. 18 ADAMTS family genes have been identified to be involved in the development and metastasis of head and neck cancer. ADAMTS14 gene polymorphisms as a risk factor for oral cancer together with environmental carcinogens (betel nut chewing and smoking) contribute to oral cancer initiation. 19 ADAMTS9 is down-regulated by promoter CpG methylation in nasopharyngeal carcinoma and inhibits angiogenesis through regulating the tumour microenvironment. 20 However, the regulatory mechanism of the ADAMTS family on OSF malignancy, a disease of oral mucosal fibrosis, remains unknown.
In this study, we explored the role of ADAMTS family members in OSF carcinogenesis and found significant differences in the expression of lncRNA ADAMTS9-AS2. LncRNA ADAMTS9-AS2 was highly expressed in normal oral mucosal tissues but down-regulated in OSF and OSCC tissues, which is associated with poor prognosis. We found that exosomes carrying ADAMTS9-AS2 inhibit cell proliferation and metastasis of OSCC cells. Mechanistically, ADAMTS9-AS2 suppresses PI3K-AKT signalling pathway and epithelial-mesenchymal transition (EMT). Our findings indicate that exosome-derived ADAMTS9-AS2 suppresses the progression of oral submucous fibrosis.

| Cell lines, tumour samples and normal tissues
Two OSCC cell lines used in this study included CAL-27 and SCC-9, purchased from the American Type Culture Collection (ATCC).
Primary fibroblast cultures of human buccal mucosa were grown and maintained according to the procedures described. 21

| Reverse transcription (RT) and quantitative real-time PCR
Total RNA was extracted using TRIzol reagent (TaKaRa) and reverse-transcribed with a Reverse Transcription System (Promega). qRT-PCRs were performed with an SYBR Green PCR Master Mix Kit (Invitrogen) on the ABI StepOne Real-Time PCR System (Applied Biosystems). The relative expression of ADAMTS9-AS2 was estimated using the threshold cycle (Ct) method, and all assays were performed in triplicate. GAPDH was used as a loading control. The primer sequences used in this study were listed as follows: ADAMTS9-AS2: 5′-CTTTAAGACCCACGAACGAC-3′ and 5′-TACTTGAGGAGAAAGCGAAA-3′; and GAPDH: 5′-TGACTT CAACAGCGACACCCA-3′ and 5′-CACCCTGTTGCTGTAGCCAAA-3′.

| Lentiviral transduction and screening of stable strains
LncRNA ADAMTS9-AS2 lentiviral expression vector was constructed by Gikai Biotechnology Co., Ltd. Lentiviral transduction was performed following the manufacturer's instructions. GFP expression was observed under a fluorescence microscope with a fluorescence rate of about 80% and cell confluence of about 80% for the lentiviral infection. Seventy two hours after infection, cells in good growth status with an infection efficiency of about 80% were screened with antibiotics for establishing stably expressing cell lines.

| Exosome isolation and purification
Exosome isolation and purification were performed using For transmission electron microscopy, exosomes were placed on a copper grid. 5-10 μL of exocrine suspension was aspirated gently onto the front of the copper-loaded mesh and carefully dried with clean filter paper after 1 minute. 10-20 μL of EM solvent was added in drops and carefully vacuumed off with a clean filter paper, and transmission electron microscopy was performed the next day. The exosomes then were quantified using NanoSight NS300 (Malvern Instruments Ltd.).

| Bisulphite treatment and promoter methylation analysis
Methylation-specific PCR (MSP) was performed as described previously. 23

| Colony formation assay
Stably transfected CAL-27 and SCC-9 cells were plated in six-well plates (400-1000 cells/well) with three replicate wells per experimental group. The inoculated cells were continued in the incubator for 14 days or until the number of cells in the majority of individual clones was greater than 50, with culture medium changes every 3 days and cell status observed. Cell clones were photographed under a fluorescence microscope. 4% paraformaldehyde was added per well, cells were fixed and PBS washed, the crystalline violet staining solution was added per well, and cells were stained for 10-20 minutes. Pictures were taken with a phase-contrast microscope, and clones were counted.

| Transwell migration and invasion assays
In vitro Transwell ® assays were performed using 24-well Transwell chambers (8-μm pore size, Corning) with or without Matrigel for cell migration and invasion, respectively. Cells were collected, resuspended in serum-free medium and added to the upper chamber (10 5 cells), and 30% FBS medium to the lower chamber. Cells were incubated at 37°C for 48 hours. The chambers were inverted on absorbent paper to remove the medium, the non-transformed cells were gently removed from the chambers with a cotton swab, and the chambers were placed in 4% paraformaldehyde fixative for 30 minutes and were stained for Giemsa (Sigma-Aldrich). Migrated cells were photographed with a random selection of the field of view using a phase-contrast microscope. The number of metastatic cells per field for each group (migratory cells per field) was counted. All experiments were independently repeated three times.

| Western blot analysis
Western blotting was performed as previously described. 25,26 Primary antibodies used in this study are as follows: AKT-total

| Statistical analysis
Data were presented as mean ± standard error of the mean (SEM).
Differences between the expression levels of lncRNAs in normal mucous, OSF and OSCC tissues were evaluated by one-way or two-way analysis of variance (ANOVA). The Kaplan-Meier survival analysis was used to evaluate the correlation of ADAMTS9-AS2 expression level with patient survival outcome. Statistical analysis was performed in excel or using SPSS 21.0 package. P < .05 was considered significant.

| Down-regulated ADAMTS9-AS2 is associated with poor prognosis of OSCC patients
Using next-generation sequencing (NGS), we previously identified the lncRNA landscape during OSF malignant progression in 2 normal mucous tissues, 8 OSF tissues with different stages and 8 OSCC combined with OSF tissues (Gene Expression Omnibus ID GSE106534). To identify the involvement of ADAMTS family members during OSF progression, 6 ADAMTS family genes were selected with rigorous criteria including fold change >4 and transcript abundance >100. Then, we chose the significantly differentially expressed transcript during OSF carcinogenesis, lncRNA ADAMTS9-AS2 as a target, which has the highest expression in normal mucous tissues, moderate expression in OSF tissues and lowest expression in OSCC combined with OSF tissues ( Figure 1A). Quantitative real-time PCR (qRT-PCR) confirmed ADAMTS9-AS2 was highly expressed in normal oral epithelium cells compared with OSF cells and OSCC cells.

| ADAMTS9-AS2 methylation mediates its reduction in OSF tumorigenesis
We next assessed the possible regulatory mechanism of ADAMTS9-AS2 reduction in OSF tumorigenesis. We firstly examined the presence of CpG island (CGI) in the ADAMTS9-AS2 promoter  Figure 2D,E). These data suggest that ADAMTS9-AS2 methylation is a tumour-specific event in the carcinogenesis of OSF. in our study. Results revealed that TSG101 and CD9 proteins were clearly expressed in exosomes extracts with proteinase K treatment alone and cell lysates, but not expressed in the supernatant and exosomes extracts with both proteinase K and Triton X-100 treatments that membranes have been permeabilized ( Figure 3C). We further investigated whether the existing pattern of extracellular ADAMTS9-AS2 in exosomes was protected from RNase A degradation. We found that RNase treatment did not change the expression levels of ADAMTS9-AS2 in exosomes too much, but its expression was significantly reduced when exosomes were treated with both RNase and Triton X-100 (***P < .001) ( Figure 3D). These data indicate that extracellular ADAMTS9-AS2 was mainly wrapped by the membrane and packaged in exosomes instead of being directly released. The exosomes secreted from OSCC cells with ADAMTS9-AS2 expression significantly inhibited OSCC cell viability (**P < .01; ***P < .001) ( Figure 4C). Moreover, the exosomes secreted by ADAMTS9-AS2-overexpressing cells (ADAMTS9-AS2-Exos) strongly suppressed the growth of both CAL-27 and SCC-9 cells to 40%-50% (*P < .05; **P < .01) by colony formation assay ( Figure 4D). These data suggest that exosomal ADAMTS9-AS2 inhibits tumour cell growth during oral tumorigenesis.

| Exosomal ADAMTS9-AS2 suppresses OSCC tumour cell metastasis, EMT and AKT signalling pathway
As some ADAMTS family members link with tumorigenesis via regulating cell migration and cell signalling pathways, the role of exosomal ADAMTS9-AS2 in OSCC cell migration and invasion abilities was further analysed by Transwell ® assays. Results showed that ADAMTS9-AS2-Exos significantly inhibited the migratory and invasive abilities of CAL-27 and SCC-9 cells (*P < .05; **P < .01) ( Figure 5A,B). We next  Figure 5C). These data suggest that exosomal ADAMTS9-AS2 suppresses tumour metastasis of OSCC, which may be through regulating EMT and AKT signalling pathways.  (Table S1). After removing low-quality reads, contaminants and adaptors, each sample clean reads were screened by small RNAs (sRNAs) within a certain length range for subsequent analysis. Because of the specificity of exosomal samples, which contain a large number of degradation fragments of other RNAs, high levels of reads fragments were concentrated at 30-32 nucleotides (nt). Length-screened sRNAs were mapped to the non-coding (nc) RNA databases. The reads identified for categories of small RNA (miRNA, rRNA, tRNA, snRNA, snoRNA, piRNA and Y_RNA) and unannotated RNAs (others) ( Figure 6A). The percentage of miRNAs in the total RNA isolated ADAMTS9-AS2 exosomes corresponded to 11.66%.

| miRNA expression profile of exosomal ADAMTS9-AS2 in OSCC cells
To identify the conserved miRNAs, all ncRNA reads from exosome libraries were compared with the specified range sequence in miRBase. A total of 736 and 798 types of known miRNAs in control and ADAMTS9-AS2 exosomes were identified, with 621 miR-NAs that were simultaneously identified in both groups ( Figure 6B, Table S2). We next analysed differentially expressed miRNAs in ADMTS9-AS2 exosomes using a twofold change and corrected level of significance (P adj < .05, q value < 0.01) as the threshold cutoff. We found that 37 miRNAs were significantly different between control and ADAMTS9-AS2 exosomes, including 23 up-regulated miRNAs and 14 down-regulated miRNAs, as shown by the Venn diagram and hierarchical clustering ( Figure 6C,D, Table S3). These results indicate the involvement of differentially expressed miRNAs in ADAMTS9-AS2-induced OSCC pathogenesis.

| Functional enrichment of target genes of miRNAs regulated by exosomal ADAMTS9-AS2
To investigate the possible functions of potential target genes of miRNAs regulated by exosomal ADAMTS9-AS2 in OSCC cells, we performed Gene Ontology (GO) and KEGG enrichment analysis on the set of target genes by differentially expressed miRNAs between control and ADAMTS9-AS2 exosomes groups (Table S4). Exosomal-transferred lncRNAs regulate apoptosis, proliferation and migration of tumour cells and induce angiogenesis, with the potential to be biomarkers for tumour diagnosis and prognosis. 28,29 LncRNA ADAMTS9-AS2 is the antisense transcript of the protein-cod- ADATMS9-AS2 contains a typical CGI and was frequently methylated in OSCC tissues, but rarely in OSF and normal mucous tissues, suggesting that its methylation is tumour-specific and could be a potential early marker for OSF and OSCC detection.

ADAMTS9-AS2-Exos
Vector-Exos Vector-Exos Vector-Exos promoter methylation also could be detected in stage I breast cancer, 36 supporting an important value of its methylation in detecting early tumours including OSCC.
Studies have demonstrated that ADAMTS9-AS2 inhibits proliferation, cell migration and invasion, and induces apoptosis in the lung, gastric and ovarian cancer cells. 30,34,37 We here found that F I G U R E 7 Functional enrichment analysis of differential miRNA target genes regulated by exosomal ADAMTS9-AS2 in OSCC cells. A, GO enrichment histogram showing functions of differential miRNA target genes in biological process, cellular component, and molecular function. B, Pathway enrichment of differential miRNA target genes by exosomal ADAMTS9-AS2 in OSCC cells. A pathway with a P-value of <.05 was defined as a significantly enriched pathway  ADAMTS9-AS2 suppressed tumour spheroid formation of gastric cells through down-regulating SPOP. 34 Further investigation of exosomal ADAMTS9-AS2 on stemness of OSF progression is needed.
In this study, we purified exosomes from the supernatant of

| CON CLUS IONS
Thus, our results indicated that exosomal ADAMTS9-AS2 was a promising novel biomarker for early detection of OSCC. Exosomal ADAMTS9-AS2 could transport to the cell microenvironment and exerts tumour-suppressive roles like exogenous ADAMTS9-AS2.
Together, our results revealed that exosomal ADAMTS9-AS2 serves as a functional mediator in cell-cell communication, which provides further evidence of the importance of exosomal lncRNAs during OSF carcinogenesis.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.